Reagents for directed biomarker signal amplification

• US 8,575,303 B2
• Filed: 01/19/2011
• Issued: 11/05/2013
• Est. Priority Date: 01/19/2010
• Status: Active Grant

• Current Assignee: Sirigen Ii Limited (Becton, Dickinson & Co)
• Original Assignee: Sirigen Group Limited (Becton, Dickinson & Co)
• Inventors: Hong, Janice W., Liang, Yongchao, Huisman, William H., Uckert, Frank Peter, Tran, Lan T., Palmer, Adrian Charles Vernon, Nguyen, Trung, Gaylord, Brent S., Baldocchi, Russell A., Wheeler, Jean M., Bartholomew, Glenn P.
• Primary Examiner(s): Truong, Duc
• Application Number:
  US13/009,764
• Publication Number:
  US 20110257374A1
• Time in Patent Office: 1,021 Days
• Field of Search: 528/371, 528/8, 528/288, 528/370, 528/425, 564/156, 435/7.1, 435/287.1, 424/9, 424/411
• US Class Current: 528/371
• CPC Class Codes: C07K 16/2812 against CD4
• CPC Class Codes: C07K 16/2815 against CD8
• CPC Class Codes: C08G 2261/1424 containing ether groups, including alkoxy
• CPC Class Codes: C08G 2261/143 Side-chains containing nitrogen
• CPC Class Codes: C08G 2261/1432 containing amide groups
• CPC Class Codes: C08G 2261/146 Side-chains containing halogens
• CPC Class Codes: C08G 2261/148 Side-chains having aromatic units
• CPC Class Codes: C08G 2261/164 comprising organic end groups
• CPC Class Codes: C08G 2261/1642 comprising reactive double bonds or triple bonds
• CPC Class Codes: C08G 2261/3142 fluorene-based, e.g. fluorene, indenofluorene, or spirobifluorene
• CPC Class Codes: C08G 2261/334 containing heteroatoms
• CPC Class Codes: C08G 2261/3422 conjugated, e.g. PPV-type
• CPC Class Codes: C08G 2261/411 Suzuki reactions
• CPC Class Codes: C08G 2261/94 Applications in sensors, e.g. biosensors
• CPC Class Codes: C08G 61/02 Macromolecular compounds containing only carbon atoms in the main chain of the macromolecule, e.g. polyxylylenes
• CPC Class Codes: C08G 61/10 only aromatic carbon atoms, e.g. polyphenylenes
• CPC Class Codes: C09B 57/00 Other synthetic dyes of known constitution
• CPC Class Codes: C09B 68/41 Polymers attached to the pigment surface C09B68/444, C09B68/446 take precedence
• CPC Class Codes: C09B 69/00 Dyes not provided for by a single group of this subclass
• CPC Class Codes: C09D 165/00 Coating compositions based on macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain C09D107/00 - C09D157/00, C09D161/00 take precedence; Coating compositions based on derivatives of such polymers
• CPC Class Codes: C09K 11/06 : containing organic luminescent materials
• CPC Class Codes: C12Q 1/6818 : involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/682 : Signal amplification
• CPC Class Codes: G01N 33/58 : involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/582 : with fluorescent label
• CPC Class Codes: H10K 85/115 : Polyfluorene; Derivatives thereof
• CPC Class Codes: H10K 85/151 : Copolymers

Abstract

Described herein are methods, compositions and articles of manufacture involving neutral conjugated polymers including methods for synthesis of neutral conjugated water-soluble polymers with linkers along the polymer main chain structure and terminal end capping units. Such polymers may serve in the fabrication of novel optoelectronic devices and in the development of highly efficient biosensors. The invention further relates to the application of these polymers in assay methods.

Claims

1. A water soluble conjugated polymer having the structure of Formula (Ia):

2. The polymer of claim 1, wherein each R is independently (CH₂)_x(OCH₂CH₂)_yOCH₃ where each x is independently an integer from 0-20, each y is independently an integer from 0 to 50, or a benzyl optionally substituted with one or more halogen, hydroxyl, C₁-C₁₂ alkoxy, or (OCH₂CH₂)_zOCH₃ where each z is independently an integer from 0 to 50.

3. The polymer of claim 1, wherein each R is (CH₂)₃(OCH₂CH₂)₁₁OCH₃.

4. The water soluble conjugated polymer of claim 1, wherein optional linkers L₁ or L₂ are selected from the group consisting of a-j having the structures:

5. The water soluble conjugated polymer of claim 1, wherein optional linkers L₁ or L₂ are

6. The water soluble conjugated polymer of claim 1, wherein G₁ and G₂ each independently have the structure

7. The water soluble conjugated polymer of claim 1, wherein G₁ and G₂ are each independently selected from the group consisting of 1-33 having the structures:

8. The water soluble conjugated polymer of claim 1, wherein the polymer contains a single conjugation site at only one terminus of the polymer chain G₁ or G₂.

9. The water soluble conjugated polymer of claim 1, wherein G₁ and G₂ is

10. The water soluble conjugated polymer of claim 1, wherein MU is selected from the group consisting of a′

-k′

having the structure;

11. The water soluble conjugated polymer of claim 1, having the structure of formula:

12. The water soluble conjugated polymer of claim 1, having the structure of formula:

13. The water soluble conjugated polymer of claim 1, having the structure of formula:

14. The water soluble conjugated polymer of claim 1, wherein the polymer further comprises a signaling chromophore selected from the group consisting of rhodamine, coumarin, xanthenes, cyanine, polymethine, pyrene, dipyrromethene borondifluoride, napthalimide, phycobiliprotein, peridinum chlorophyll protein, conjugates thereof, and combinations thereof.

15. The water soluble conjugated polymer of claim 1, wherein the polymer is conjugated to a secondary dye reporter and an antibody.

16. The water soluble conjugated polymer of claim 5, wherein the linker,

17. The water soluble conjugated polymer of claim 1, wherein the polymer is conjugated to a streptavidin, antibody or nucleic acid and used as a direct fluorescent reporter.

18. The water soluble conjugated polymer of claim 1, wherein the polymer is conjugated to thiol groups at the hinge region of an antibody.

19. The water soluble conjugated polymer of claim 1, wherein the polymer is conjugated to an amine group on a protein which is modified with a heterobifunctional linker.

20. The water soluble conjugated polymer of claim 1, wherein the polymer is conjugated to an antibody.

21. The water soluble conjugated polymer of claim 20, wherein the polymer and antibody excited at about 405 nm in a flow cytometry assay where the specific signal is at least 3 fold greater than the same antibody conjugated to Pacific Blue.

22. The water soluble conjugated polymer of claim 1, wherein the polymer is ≧

95% pure.

23. The water soluble conjugated polymer of claim 1, wherein the polymer is used in flow cytometry assays to identify different cell markers or cell types.

24. The water soluble conjugated polymer of claim 1, wherein the polymer is used for intracellular staining.

25. The water soluble conjugated polymer of claim 1, comprising a minimum number average molecular weight of greater than 40,000 g/mol and a water solubility of greater than 50 mg/mL in pure water or a phosphate buffered saline solution.

26. The water soluble conjugated polymer of claim 1, comprising at least two unique conjugation linkers which can conjugated to two unique materials.

34. The water soluble conjugated polymer of claim 1, wherein the polymer is conjugated to an amine group on a protein.

35. The water soluble conjugated polymer of claim 1, wherein optional linkers L₁ or L₂ have the structure:

36. The water soluble conjugated polymer of claim 1, wherein a is from about 40% to 60%.

37. The water soluble conjugated polymer of claim 1, wherein b is from about 40% to 60%.

38. The water soluble conjugated polymer of claim 1, wherein the polymer has the structure:

39. The water soluble conjugated polymer of claim 1, wherein the polymer has the structure:

40. The water soluble conjugated polymer of claim 35, wherein optional linkers L₁ or L₂ are selected from the group consisting of

41. The water soluble conjugated polymer of claim 1, wherein MU is selected from the group consisting of

27. A conjugated polymer complex comprising:

a polymer coupled to at least one biomolecule selected from the group consisting of a sensor biomolecule, a bioconjugate and a target biomolecule wherein the polymer is covalently bound by at least one bioconjugation site pendant thereto, and the polymer comprises a signaling chromophore or a signaling chromophore optionally is covalently bound to the polymer or the sensor biomolecule;

wherein the polymer comprises the structure of formula;

28. The conjugated polymer complex of claim 27, wherein the sensor biomolecule is selected from the group consisting of an avidin, streptavidin, neutravidin, avidinDN, and avidinD.

29. The conjugated polymer complex of claim 27, further configured to bind to a complex selected from the group consisting of a biotin-labeled antibody, biotin-labeled protein, and biotin-labeled target biomolecule.

30. The conjugated polymer complex of claim 27, wherein the sensor biomolecule is an antibody.

31. The conjugated polymer complex of claim 27, wherein both the signaling chromophore and the sensor biomolecule are covalently linked to the multichromophore through a plurality of linkers.

32. The conjugated polymer complex of claim 27, wherein the signaling chromophore, when covalently bound to the polymer or the sensor biomolecule, is an organic dye.

33. A water soluble conjugated polymer having the structure of Formula (Ia):